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A New Species of Amaurobius C.L. Koch, 1837 (Araneae: Amaurobiidae) from Apennine Mountains (Italy) with the Description of the Male of A

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A New Species of Amaurobius C.L. Koch, 1837 (Araneae: Amaurobiidae) from Apennine Mountains (Italy) with the Description of the Male of A Zootaxa 4276 (4): 479–502 ISSN 1175-5326 (print edition) http://www.mapress.com/j/zt/ Article ZOOTAXA Copyright © 2017 Magnolia Press ISSN 1175-5334 (online edition) https://doi.org/10.11646/zootaxa.4276.4.2 http://zoobank.org/urn:lsid:zoobank.org:pub:E75ABE24-5B70-42E0-A240-29187F0D4140 A new species of Amaurobius C.L. Koch, 1837 (Araneae: Amaurobiidae) from Apennine Mountains (Italy) with the description of the male of A. pavesii Pesarini, 1991 FRANCESCO BALLARIN1,2,3,5 & PAOLO PANTINI4 1Institute of Zoology, Chinese Academy of Sciences (IOZCAS), 1 Beichen West Road, Chaoyang District, Beijing 100101, P. R. China. E-mail: [email protected] 2University of Chinese Academy of Sciences, Beijing, 100049, China 3Sezione di Zoologia, Museo Civico di Storia Naturale di Verona, Lungadige Porta Vittoria, 9, I-37129 Verona, Italy 4Museo Civico di Scienze Naturali "E. Caffi" di Bergamo. Piazza Cittadella, 10, I-24129 Bergamo, Italy. E-mail: [email protected] 5Corresponding author Abstract A new species of the genus Amaurobius C.L. Koch, 1837, Amaurobius pesarinii sp. n., from the Apennine Mountains (Italy) is described on the basis of both sexes. The male of A. pavesii Pesarini, 1991, previously unknown, is also de- scribed. Females of A. pavesii and both sexes of the similar A. scopolii Thorell, 1871 are re-described. Detailed description of morphological characters allow clear separation between these species and the closely related A. ferox (Walckenaer, 1820). Their phylogenetic relationships among the most common Italian Amaurobius species are discussed on the basis of molecular data (partial fragments of the genes COI, 16S and H3), together with their distribution across the Italian Pen- insula. This is the first contribution to a multi-locus phylogenetic tree of European Amaurobius species. The synonymy of A. sciakyi Pesarini, 1991 with A. ruffoi Thaler, 1990 is proposed based on morphological characters. Key words: Arachnida, taxonomy, phylogenetic analysis, DNA Introduction Amaurobius C.L. Koch, 1837, is the nominal genus of the family Amaurobiidae. It contains 64 mostly Holarctic known species (World Spider Catalog 2016). The European fauna includes 36 species (Nentwig et al. 2016), 11 of which occur in Italy: A. crassipalpis Canestrini & Pavesi, 1870, A. erberi (Keyserling, 1863), A. fenestralis (Ström, 1768), A. ferox (Walckenaer, 1830), A. jugorum L. Koch, 1868, A. obustus L. Koch, 1868, A. pallidus L. Koch, 1868, A. pavesii Pesarini, 1991, A. ruffoi Thaler, 1990, A. scopolii Thorell, 1871, and A. similis (Blackwall, 1861) (Pantini & Isaia 2016). So far, few articles have been published on the Italian amaurobiid fauna. The majority of these species remain poorly studied, and their ecology and distribution on the Italian peninsula are little understood. Furthermore, the morphological similarities between females of closely related species, and the lack of reliable diagnostic characters, have likely led to possible misidentifications, further confounding the taxonomy of the group. In his publication on the Amaurobiidae of northern Italy, Pesarini (1991) analyzed the morphology of seven Italian Amaurobius species, including A. scopolii. He also described two new spiders: A. sciakyi from the Brescia and the Venetian Prealps, and A. pavesii (based only on female specimens) from the Apuan Alps. The morphology and distribution of A. sciakyi closely resemble those of A. ruffoi, and the two species are treated as conspecific by Italian arachnologists; however, their synonymy has never been formalized (Trotta 2005, Pantini & Isaia 2016). A. pavesii and A. scopolii are morphologically similar, although they can be distinguished by the different shape of the epigyne (Pesarini 1991). The male of A. pavesii has not yet been described. While examining Amaurobius material from Italy, we compared numerous specimens of A. ruffoi with the type material of A. sciakyi, confirming their synonymisation based on morphology and distribution. Furthermore, we Accepted by P. Jaeger: 10 Apr. 2017; published: 14 Jun. 2017 479 found several specimens of both sexes belonging to A. pavesii and A. scopolii. A careful examination of the samples led to the identification of the undescribed male of A. pavesii. Our observations also revealed new, detailed morphological characters in the palp, epigyne and vulva which enabled a clear separation of the two species and the closely related A. ferox. Furthermore, the material showed the presence of a new, closely related species distributed in the central Apennine mountain chain. Presumably, this new species had previously been confused with A. scopolii. This paper aims to describe the newly discovered species, and the undescribed male of A. pavesii providing detailed information on their identification. Few molecular studies have involved species of the genus Amaurobius, evident in the scarcity of sequences of amaurobiids available from public online datasets such as GenBank (GenBank 2016). So far, no specific studies on Amaurobius phylogenetics have been conducted using molecular analyses, the relationships between the European species remain unresolved. In order to genetically confirm the newly discovered species, and to shed light on its phylogenetic position, a molecular phylogenetic analysis was conducted. Our phylogeny was based on mitochondrial and nuclear gene fragments involving the most common Italian Amaurobius species. This is the first contribution to a multi-locus phylogenetic tree addressing relationships among European Amaurobius. Material and methods Taxonomy. A Wild M5A stereomicroscope with an ocular micrometer was used for examining and measuring specimens at the Museo Civico di Storia Naturale di Verona, Italy. Photographs of Amaurobius pesarinii sp. n. and A. scopolii were made using an Olympus Camedia E-520 digital camera mounted on an Olympus SZX16 stereomicroscope at the Zoological Museum, University of Turku, Finland. Pictures of A. ferox and A. pavesii were taken with an Olympus c7070 wide zoom digital camera mounted on an Olympus BX51 compound microscope at the Institute of Zoology, Chinese Academy of Sciences, Beijing, China. Images were combined using CombineZP image stacking software and subsequently processed with the program Photoshop CS6. To adequately see the vulva, epigynes were dissected with a needle and macerated in 10% KOH. All the specimens used for morphological analysis are preserved in 70% denatured alcohol and stored in the collections of the following institutions: MLSF=Museo di Storia Naturale, sezione di Zoologia "La Specola", Firenze, MSNB=Museo Civico di Scienze Naturali "E. Caffi" di Bergamo, MSNM=Museo Civico di Storia Naturale di Milano, MSNV=Museo Civico di Storia Naturale di Verona, ULSR=Dipartimento di Biologia Animale e dell'Uomo, Università degli Studi di Roma "La Sapienza", Italy and ZMUC=Zoological Museum, University of Copenhagen, Denmark. Leg measurements are shown as: total length (femur, patella, tibia, metatarsus, tarsus). Leg spination is reported from proximal to distal position, the possible variation in number of spines present in some of the specimens is provided in parentheses. All measurements in the text are given in millimeters. The following abbreviations are used: AM=anterior median eyes; Cd=copulatory ducts; Co=conductor; d=dorsal spines; Da=dorsal tibial apophysis; Em=embolus; Fd=fertilization duct; Ib=epigynal median plate internal bump; Im=interior margins of the epigynal median plate; Ll=lateral lobe; Ma=median apophysis; Mp=epigynal median plate; p=prolateral spines; PM=posterior median eyes; r=retrolateral spines; RTA=retrolateral tibial apophysis; Sp=spermatheca; Ta=tegular apophysis; Te=tegulum; v=ventral spines; Ve=ventral edge of the median plate. Molecular analysis and related sample collection and storage. Representative samples of the most common Italian Amaurobius species (9 species) were collected across the Italian Peninsula, with the exception of A. pallidus and one specimen of A. fenestralis, which were collected in Romania. Additional sequences were obtained from GenBank and BOLD online databases (see Appendix 1 for the detailed localities and accession codes). Live specimens were collected by hand in the field, immediately put in 95% pure ethanol, and subsequently stored at - 20°C in the Institute of Zoology, Chinese Academy of Sciences, Beijing, China (IZCAS) after the morphological identification of the species. Total genomic DNA was extracted from a single leg of an adult specimens using a TIANamp Genomic DNA Kit (TIANGEN) following the standard manufacturer’s protocol. Amaurobius has been traditionally considered closely related to Callobius, and recent molecular studies appear to confirm this affinity (Miller et al. 2010). Two species of Callobius, C. claustrarius (Hahn, 1833) and C. sp. were therefore established as outgroups to root the tree using a sample from Romania (C. claustrarius) and sequences downloaded from GenBank (C. sp., accessions: FJ607559, FJ607450, FJ607598). 480 · Zootaxa 4276 (4) © 2017 Magnolia Press BALLARIN & PANTINI PCR amplification and sequencing. Partial fragments of the mitochondrial genes cytochrome c oxidase subunit I (COI) and 16S rRNA (16S) and the nuclear gene Histone 3 (H3) were selectively amplified using the primers and the protocols as reported in Appendix 2. The PCR amplifications were performed with an Eppendorf thermal cycler (Hamburg, Germany) using a final volume of 25µl. Purified PCR products were sequenced in both directions on an ABI 377 automatic
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